CN105177160A - Primers for detecting plurality of newborn inherited metabolic disease causing genes and kit - Google Patents
Primers for detecting plurality of newborn inherited metabolic disease causing genes and kit Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention discloses a design way of primers for in-vitro qualitative detection of newborn inherited metabolic disease genes. The design way comprises the following steps: (1) acquiring relevant disease-causing gene information of inherited diseases: acquiring the relevant disease-causing gene information, including gene location information and mutation site information of a plurality of inherited diseases through an OMIM (Online Mendelian Inheritance in Man) database, and acquiring exon and intron information of relevant genes from an NCBI (National Center for Biotechnology Information) database according to the obtained gene information; (2) designing primers specific to the relevant genes: designing corresponding specific primers F1 and R1 specific to an exon and an exon-intron combination area of each gene. The invention also discloses the first-round PCR (Polymerase Chain Reaction) primer sequence list of syndromic deafness, a first-round PCR primer list of G6PD genes, and a first-round PCR primer sequence list of SLC12A3 genes. The primers can be used for detecting relevant genes of 22 types of common newborn inherited diseases.
Description
Technical field
The present invention relates to molecular biology and medical science, particularly a kind of newborn infant's Inherited Metabolic Disorders gene tester that can be used for two generations order-checking platform.
Background technology
Inherited metabolic disease (inheritedmetabolicdiseases, IMD) Organic acidemia (inbornerrorsofmetabolism is also called, IEM) be because the encoding gene maintaining body eubolism certain enzyme necessary, transporter, film or acceptor etc. is undergone mutation, the product function making it encode changes, and occurs a class disease of corresponding laboratory examination exception and clinical symptom.It relates to the exception of the many kinds of substance metabolism such as amino acid, organic acid, lipid acid, ornithine cycle, carbohydrate, lysosome, steroid.Such disease is mostly single gene inheritance disease, and minority is chondriogen inherited disease.Normal after this kind of disease incidence is that Progressive symmetric erythrokeratodermia increases the weight of, and as failed EARLY RECOGNITION diagnosis, intervention, treatment, often causes dying young in early days or life-long disabilities, and is maximum with neural, intelligent disability, can have a strong impact on population quality, cause family and social heavy burden.Therefore carry out the early screening of IEM, realize the key that early diagnosis becomes IMD control, avoid and reduce the generation of handicapped child, to improving the health of the people, promoting the national economic development and having great significance.
At present, the diagnostic method of IMD mainly contains urine chromogenic assay, Bacillus subtilus inhibition test, enzyme assay, Gas chromatographyMass spectrometry (GC-MS), tandem mass spectrum coupling technique (MS/MS), single gene analyses etc.These diagnostic methods are used for IMD examination and have limitation: (1) general flux is low, generally once can only detect a kind of to tens kinds, can not meet IMD and can detect increasing year by year of disease kind, and need there is anticipation accurately to inherited metabolic disease; (2) detection method of different I MD is different, and sample is various, and then detect gene level as some diseases detects other diseases of protein level, some disease needs urine specimen, and other then need blood sample.Therefore the little applicable examination carrying out large-scale inherited metabolic disease with this, needs the technology wanting a kind of energy high throughput testing IMD various diseases badly.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of efficient detection method of common newborn infant's Inherited Metabolic Disorders genes involved, and detection method of the present invention is intended to reduce testing cost.
In order to solve the problems of the technologies described above, the invention provides a kind of design of the primer for the outer qualitative detection of newborn infant's inherited metabolic disease genosome, comprising the steps:
1) the relevant Disease-causing gene information of inherited disease, is obtained:
The relevant Disease-causing gene information of multiple (being such as 22 kinds) heredopathia is obtained by omim database, comprise gene location information, mutational site information, then obtain exon and the intron information of genes involved according to the gene information obtained from ncbi database;
2), for genes involved design primer:
For the exon of each gene and Auele Specific Primer F1 and R1 of exon intron calmodulin binding domain CaM design correspondence, principle of design is: each exon extension 5bp, to ensure exon intron calmodulin binding domain CaM to be included; Primer sequence district guard, namely do not exist sudden change or SNP, Tm value homogeneous, the difference of Tm value is within 5 DEG C; Amplified production has the overlap of 10bp at least; Before often pair of primer, add the sequence A 1 of the preceding paragraph designed, designed and the recognition sequence of A2 and a section of 6 bases, in order to two generation sequencing result identification; The sequence composition diagram of primers F 1 and R1 is shown in schematic diagram, Fig. 1;
Remarks illustrate: that what A1 was corresponding is F1, A2 is corresponding is R1.
The different exons that can be formed by variable sheer due to a lot of gene combine the different transcripts formed, montage becomes the exon of each transcript also different in quantity or size, the exon of the gene related in the present invention is the template designing primer by the exon set of the different transcripts found in current NCBI, and principle is shown in Fig. 2.
Each expanding fragment length is about 300bp.
The sequence of A1 and A2 is as follows:
A1(5’-3’)：TCTTTCCCTACCGACGCTCTTCCGATCT
A2(5’-3’)：GTGACTGGAGTCAGACGTGTGCTCTTCCGATCT
Recognition sequence has 12 (selecting a use), is respectively ATCACG, TTAGGC, ACAGTG, TGACCA, GCCAAT, CAGATC, ACTTGA, ATCACG, TAGCTT, GGCTAC, CTTGTA, GATCAG, every section of recognition sequence can be added in F1 and R1 at random, and in F1 and R1, the array mode of recognition sequence can as the recognition signal of sample in sequencing result and gene.
The present invention also provides the primer for the outer qualitative detection of newborn infant's inherited metabolic disease genosome simultaneously: the first round PCR primer sequence table of syndromic deafness is as following table 1; The first round PCR primer sequence table of G6PD gene is as following table 2; The sequence table of SLC12A3 gene first round PCR primer is as following table 3;
Table 1
Table 2
Table 3
The present invention also provides a kind of test kit simultaneously, and this test kit contains the primer described in above-mentioned table 1 ~ table 3, and this test kit can detect multiple neonatal hereditary diease simultaneously.
Specifically, this test kit may be used for detecting pku, argininemia, arginyluccinicaciduria, citrullinemia I type, citrullinemia II type, maple syrup urine disease, tyrosinemia I type, tyrosinemia II type, tyrosinemia type III, Ornithine carbamoyltransferase deficiency disease, galactosemia, glucose 6 phosphate dehydrogenase deficiency, isovaleric acidemia, propionic acidemia, glutaric acidemia I type, carnitine transit barrier, Carnitine palmitoyltransferase I deficiency disease, Carnitine palmitoyltransferase II deficiency disease, heredity nonsyndromic sensorineural, the gene that these 22 kinds of common neonatal hereditary diease of Gitelman syndrome are relevant.
The present invention also provides the method for the outer qualitative detection of a kind of newborn infant's inherited metabolic disease genosome simultaneously.
The solution of the present invention is specific as follows:
1, the relevant Disease-causing gene information (the same) of inherited disease is obtained;
2, for genes involved design primer (the same),
3, pcr amplification object fragment:
With the genomic dna of people for template, initial amount is 20ng, carries out pcr amplification, obtains two ends respectively with the object fragment of A1 and A2 machine recognition sequence, and carries out purifying, obtains first time PCR primer P1.
4, take P1 as template, carry out second and take turns pcr amplification.
The sequence of taking turns the primers F 2 of PCR and R2, F2 and R2 for primary PCR primer P1 design second is as follows:
F2(5’-3’)：
ATGATACGGCGACCACCGGATCTACACTCTTTCCCTACACGACGCTC
R2(5’-3’)：
CAGCAGAAGACGGCATACGAGTCGTGATGTGACTGGAGTTCAGACGTG
Take P1 as template, initial amount is 50ng, carries out second and takes turns pcr amplification, obtain product P 2, carry out purifying to P2, namely obtains and directly can carry out with Illumina bis-generation order-checking platform the library L that checks order.
Utilize method of the present invention only to carry out sequencing library that two-wheeled pcr amplification just can obtain exon that Disease-causing gene is correlated with, exon intron land sequence rapidly, Illumina order-checking platform directly can be utilized to carry out high-flux sequence, for multiple exons of a multiple gene of sample, can by after carrying out balanced mix after first round PCR primer purifying, carry out second as a template and take turns pcr amplification, greatly save operating time and expense.
The schematic diagram of the method as depicted in figs. 1 and 2.
Method of the present invention may be used for detecting pku, argininemia, arginyluccinicaciduria, citrullinemia I type, citrullinemia II type, maple syrup urine disease, tyrosinemia I type, tyrosinemia II type, tyrosinemia type III, Ornithine carbamoyltransferase deficiency disease, galactosemia, glucose 6 phosphate dehydrogenase deficiency, isovaleric acidemia, propionic acidemia, glutaric acidemia I type, carnitine transit barrier, Carnitine palmitoyltransferase I deficiency disease, Carnitine palmitoyltransferase II deficiency disease, heredity nonsyndromic sensorineural, the gene (as shown in table 4) that 22 kinds of common neonatal hereditary diease such as Gitelman syndrome are relevant, this test kit can carry out the order-checking of two generations and analysis to the sequence of the exon of 40 Disease-causing genes that 22 kinds of common newborn infant's Inherited Metabolic Disorders are correlated with and exon intron calmodulin binding domain CaM, applying this test kit can the abrupt information of detection genes involved of precise and high efficiency, realizes the efficient detection to multiple neonatal hereditary diease, and greatly can reduce testing cost.
Common newborn infant's Inherited Metabolic Disorders that this test kit comprises and genes involved are in table 4:
The gene information table of table 422 kind of common neonatal hereditary diease
In sum, the present invention is directed to common and newborn infant's hereditary metabolic disorders that hazardness is higher, as pku, argininemia, maple syrup urine disease, Ornithine carbamoyltransferase deficiency disease, galactosemia, glucose 6 phosphate dehydrogenase deficiency, isovaleric acidemia, heredity nonsyndromic sensorineural, 22 kinds of common inherited diseases such as Gitelman syndrome, for the corresponding primer of common causative gene design, by multiplexed PCR amplification exon, exon intron calmodulin binding domain CaM, adopt amplicon sequence measurement, can high throughput testing heredopathia, in order to fast, easy, examination heredopathia efficiently, realize early diagnosis and the early treatment of disease, reduce lethality rate and the disability rate of inherited disease, alleviate the burden of family and society.
The present invention has following advantage and innovative point:
1. advantage: disease gene mutated site is not fixing, therefore cannot detect by taqman probe method, must use sequencing.At present, generation order-checking can only detect 500-1000bp length at every turn, if gene reaches hundreds of kb, it is very expensive that detection can become, and it is very long to detect spended time.Directly utilize full-length genome two generation to check order or full exon order-checking, then high cost, simultaneously useless in a large number sequencing result, what analysis also can be made to become is very difficult.The present invention only carries out amplicon order-checking for the Disease-causing gene exon of some Inherited Metabolic Disorders and exon intron land, enormously simplify trace routine, and makes subsequent analysis simpler, can obtain result rapidly.
2. innovative point: the present invention only needs to carry out twice PCR amplification, just the exon region of genes involved can be increased out, its product can be directly used in the order-checking of two generations, for in this process first time pcr amplification design of primers compare with common PCR primer, add the identifier of one section of special sequence and one section of 6 base in the primer front end of target fragment, there is novelty; The present invention merges the exon region of the different transcripts of gene first in addition, exon set after merging is carried out design of primers as the template of this gene extron, more fully cover the exon region of this gene, more can find the mutational site of exon region, make result more comprehensively, more credible.
Remarks illustrate: None-identified in countless sequencing sequences can be caused to go out the sequence wanted required for the present invention if remove " identifier " of the present invention; That is, the present invention cannot be realized.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Gene extron amplification schematic diagram for the purpose of Fig. 1;
Fig. 2 is the exon aggregate manner schematic diagram of certain gene 3 transcripts.
Fig. 3 is SLC26A4 gene Sanger sequencing result; Arrow shows c.919-2T>C homozygous mutation.
In Fig. 3, upper figure is standard sequence (wild-type), and figure below is clinical samples sequencing result, and arrow represents homozygous mutation.
Fig. 4 is GJB2 gene Sanger sequencing result; C.109G>A arrow shows (p.Val37Ile) heterozygous mutant.
In Fig. 4, upper figure is standard sequence (wild-type), and figure below is clinical samples sequencing result, and arrow represents heterozygous mutant.
Fig. 5 is the Sanger sequencing result of G6PD gene.Upper figure is the reverse sequence (wild-type) of normal people, and figure below is clinical samples backward sequencing result, and arrow place is homozygous mutation c.678C>G.
Fig. 6 is the Sanger sequencing result of SLC12A3 gene.Upper figure is normal people's sequence (wild-type), and figure below is clinical samples sequencing result, and arrow place is phase shift mutation section start, and genotype is loss of heterozygosity.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.
Embodiment 1: the present embodiment have chosen non-syndromic cleft lip and palate related genes GJB2, GJB3, SLC26A4,12SrRNA, COCH and DFNA5 totally six genes,
One, sample prepares (acquisition of sequencing library)
Carrying out two-wheeled pcr amplification to the exon of this gene and exon intron land, to obtain the step of sequencing library as follows:
1. the acquisition of Disease-causing gene information.
In the omim database of OMIM (OnlineMendelianInheritanceinMan) website or NCBI, carry out the search of non-syndromic cleft lip and palate, obtain relative focus Disease-causing gene, obtain relevant mutational site information.
2. in NCBI, search for the exon of each related genes and the sequence of intron and positional information.
3. multiple PCR primer Design and synthesis
Exon for above gene designs corresponding PCR primer, according to previous designs principle, devises 50 pairs of primers altogether, and the primer of all pcr amplifications entrusts the synthesis of prompt base (Shanghai) trade Co., Ltd in the English Weihe River, HPLC purifying, and concrete primer sequence is in table 2:
The PCR primer sequence table of table 1 first round
Note: in table, the base sequence of six lowercases is recognition sequence.
4. the extraction of genomic dna
Blood sample is from hospital of Zhejiang Province, clinical diagnosis is non-syndromic cleft lip and palate, blood sample 200 μ l, with QIAGENDNeasyBlood & TissueKit (250) test kit, (article No.: the extraction 69506) carrying out genomic dna, it is 22ng/ μ l that Nanodrop1000 surveys concentration.
First round pcr amplification.
More than 5.1 to extract the genomic dna obtained be template (initial amount is 30ng), with 50 of table 2 pairs of primer sequences for two ends primer, carries out first round pcr amplification respectively, thus obtain 50 amplified productions respectively, respectively called after P1-1 ~ P1-50.
The system of 5.2 epicycle pcr amplifications is as follows:
* Q5DNA polysaccharase is purchased from the Q5 amplification kit of NEB, article No.: E0555L;
* 5 × Q5reactionBuffer, article No. B9027S;
* dNTPMixture is purchased from Takara company, and article No. is 4019.
PCR reaction tubes is put in PCR instrument by 5.3, runs program shown in following table:
After more than reaction terminates, the PCR primer obtained is carried out purifying with magnetic bead respectively, gets 5ulPCR product 1% gel electrophoresis analysis PCR primer clip size, measure PCR primer concentration.
6 second take turns pcr amplification
50 PCR primer obtained in 6.1 steps 5.3 obtain mixed solution after carrying out equivalent (each product 30ng) mixing, get 50ng takes turns pcr amplification template as second, take turns the primer of PCR using F2 and R2 as second.
6.2PCR reaction system is as follows:
* Q5DNA polysaccharase is purchased from the Q5 amplification kit of NEB, article No.: E0555L;
* dNTPMixture is purchased from Takara company, and article No. is 4019.
PCR reaction tubes is put in PCR instrument by 6.3, runs program shown in following table:
After more than reaction terminates, by the PCR primer QIAquickPCR purification kit (QIAGEN) obtained, according to operation instructions purifying amplified production, namely this product can be used for Illumina order-checking platform and carries out upper machine order-checking.
7 carry out machine on Illumina by standard program.
Two, interpretation of result
1. the raw data of pair IlluminaMiseq sequenator generation, adopts BWA alignment algorithm software to carry out original long sequence (reads) of reading and carries out preliminary comparison with reference to genome.
2. comparison processes with recalibration again
Adopt the instrument that three kinds important respectively: (1) base quality score is corrected again; (2) indel close region local again matching ratio to process; (3) shielding repeats to read long sequence.Shielding repeats to check order, and to read long processing intent be consider that process to be checked order the sequence redundancy caused by PCR that both-end exists at pair-end, simultaneously for the treatment of repeat to read long in indel region obtain border to there is the situation (1) of Different matching high efficiency with known site (indel, SNP) again mate and comparison process, can operate low cover degree region (only known indel being mated again).(2) adopt base mispairing mode in order to judge that site is the need of matching ratio pair again, and carry out comprehensively local matching treatment again.
3. subordinate phase (phase2) variant recognition and genotype calculate, the merging treatment that the sequence of first again mating for rectification compares result, carries out merging and is used for follow-up varient and genotypic identification by the comparison result of all genome sequencings.
4. multiplicity sampling SNP and Indel identifies and adopts the universal genetic type recognizer based on Bayes's genotype likelihood ratio model algorithm to identify SNP and indel.The principle of Bayes's genotype likelihood ratio model algorithm is the genotype and gene frequency that in the colony or independent part sample simultaneously estimating to comprise N number of sample at, most probable occurs, and the varient accurately calculated on each nucleotide position and genotypic posterior probability.Universal genetic type recognizer is used for identifying statistically the judged allelic varient of non-reference sequence (SNP, Indel) position and genotype.
5. carry out machine on IlluminaMiseq by standard program, output is about 150M data volume.
6. data analysis
According to the analysis of biological information technology of high-flux sequence routine, the patient's sample obtained sudden change (only listing deaf-related gene) as shown in the table:
Note: Freq_dbSNP represents the frequency of this sudden change in dbSNP database; Freq_HapMap represents the frequency of this sudden change in dbSNP database; Freq_1kgenome represents the frequency of this sudden change in dbSNP database; * represents pathogenic mutation.
Interpretation of result: inquiry in omim database (http://omim.org), find in this patient's sample, have two genes to there is pathogenic mutation, as in table, * * represents, on SLC26A4 gene, (Intron7) finds that c.919-2T>C 1 sheared sudden change, is homozygote; Existing pertinent literature reports that it is pathogenic, and its probability occurred in crowd is lower.SLC26A4 gene is Pendred syndrome (Pendredsyndrome; And autosomal recessive deafness companion Large Vestibular Aqueduct disease (Deafness, autosomalrecessive4, withenlargedvestibularaqueduct No. OMIM: #274600); Disease-causing gene, is autosomal recessive inheritance No. OMIM: #600791).Finding 1 missense mutation c.109G>A (p.Val37Ile) in its GJB2 gene coding region (EX2/CDS1), is heterozygote; Existing pertinent literature reports that it is pathogenic.GJB2 is the deaf 1A type of autosomal recessive (Deafness, AutosomalRecessive1A, and the Disease-causing gene of deaf 3A type (Deafness, AutosomalDominant3A, OMIM:#601544) of autosomal dominant OMIM:#220290).Non-syndromic cleft lip and palate genes involved also finds multiple shearing, missense or synonym variation.Therefore, the shearing sudden change inferring SLC26A4 gene c.919-2T>C with the missense mutation of GJB2 gene c.109G>A (p.Val37Ile) suspicious disease cause mutation that is deaf patient.
C.919-2T>C the sudden change gold standard Sanger method of wherein SLC26A4 gene is verified, finds the mutation type completely the same (as shown in Figure 3) detected with the present invention.In like manner, GJB2 gene Sanger sequencing result, c.109G>A arrow shows (p.Val37Ile) heterozygous mutant.The mutation type detected with the present invention completely the same (as shown in Figure 4).
Above result display, utilize method of the present invention can carry out the detection of Inherited Metabolic Disorders pathogenic mutation based on two generations order-checking platform, result accurate and effective, provides strong theoretical direction for doctor treats targetedly to heredity metabolize patient further.
Remarks illustrate: omim database has informed the mutant gene locus information reported with disease-related.
Through the analysis of two generations order-checking, we only find that GJB2 gene and SLC26A4 gene exist confirmed pathogenic mutation, and the sudden change of other genes is all non-pathogenic mutation, therefore only needs checking pathogenic mutation site being carried out to generation sequencing.
Embodiment 2: the method for reference example 1, to certain glucose 6 phosphate dehydrogenase deficiency patient, utilize method design primer of the present invention, first round primer sequence is as following table 2:
Table 2, the sequence table of G6PD gene first round PCR primer
After first round PCR completes, carry out second take turns PCR after high-flux sequence (concrete steps and method are shown in example 1), the result obtained is as follows:
The high-flux sequence result of G6PD deficiency disease patient is:
C.678C>G the mutational site gold standard Sanger method of wherein G6PD gene is verified (with normal artificial contrast), finds that the mutation type detected with the present invention is completely the same, as shown in Figure 5:
Fig. 5 is the Sanger sequencing result of G6PD gene.Upper figure is the reverse sequence (wild-type) of normal people, and figure below is clinical samples backward sequencing result, and arrow place is homozygous mutation c.678C>G.
Do not find that this patient exists other known or doubtful disease cause mutations within the scope of Product checking.State in conjunction with frequency in each database of the sickness rate of disease, site and the clinical master of person under inspection, detect 1 not bright site of clinical meaning.The not bright site of clinical meaning is not enough to the generation explaining disease, only as doctor's reference.
Site details: G6PD; NM_000402; C.678C>G; P.Ile226Met; CDS6; Hemi: missense mutation, temporarily without the pertinent literature report that this site is pathogenic, clinical meaning is not bright.This site occurrence frequency in crowd is extremely low.Carry out protein function prediction through SIFT and Polyphen to it, result is harmful.
Above result display, utilizes method of the present invention can detect the detrimental mutation site of G6PD gene, provides strong theoretical direction for doctor treats targetedly to heredity metabolize patient further.
Example 3: the method for reference example 1, to the newborn infant of certain Gitelman syndrome patient, method of the present invention is utilized to carry out high-flux sequence after pcr amplification to related genes SLC12A3 gene design primer (primer of first round PCR is as table 3), judge whether the son of this patient suffers from Gitelman syndrome, obtains following result:
Table 3, the sequence table of SLC12A3 gene first round PCR primer
The neonatal high-flux sequence result of Gitelman syndrome patient is:
C.176_179delACAAA the deletion mutantion site gold standard Sanger method of wherein SLC12A3 gene is verified, find that the mutation type detected with the present invention is completely the same, after having lacked ACAAA sequence, cause phase shift mutation, it is heterozygote simultaneously, cause follow-up bimodal (arrow instruction place starts frameshit occurs), as shown in Figure 6.
Fig. 6 is the Sanger sequencing result of SLC12A3 gene.Upper figure is normal people's sequence (wild-type), and figure below is clinical samples sequencing result, and arrow place is phase shift mutation section start, and genotype is loss of heterozygosity.
C.176_179delACAAA, this lacks and does not all occur in the databases such as dbSNP, for new deletion mutantion, this disappearance causes follow-up amino acid frameshit, and this sports loss of heterozygosity, concerning this autosomal recessive is sick, this newborn infant is Disease-causing gene carrier.This result can as doctor's clinical reference.
Result shows, and probe of the present invention can be used for new-generation sequencing, detects the mutational site of heredity metabolize patient, provides theoretical direction for doctor carries out immunotherapy targeted autoantibody to heredity metabolize patient further.
Finally, it is also to be noted that what enumerate above is only a specific embodiment of the present invention, the invention is not restricted to above embodiment, various deformation perhaps can also be had.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (4)
1., for the design of the primer of the outer qualitative detection of newborn infant's inherited metabolic disease genosome, it is characterized in that comprising the steps:
1) the relevant Disease-causing gene information of inherited disease, is obtained:
Obtain the relevant Disease-causing gene information of multiple heredopathia by omim database, comprise gene location information, mutational site information, then obtain exon and the intron information of genes involved according to the gene information obtained from ncbi database;
2), for genes involved design primer:
For the exon of each gene and Auele Specific Primer F1 and R1 of exon intron calmodulin binding domain CaM design correspondence, principle of design is: each exon extension 5bp, to ensure exon intron calmodulin binding domain CaM to be included; Primer sequence district guard, namely do not exist sudden change or SNP, Tm value homogeneous, the difference of Tm value is within 5 DEG C; Amplified production has the overlap of 10bp at least; Before often pair of primer, add the sequence A 1 of the preceding paragraph designed, designed and the recognition sequence of A2 and a section of 6 bases, in order to two generation sequencing result identification;
The sequence of A1 and A2 is as follows:
A1(5’-3’)：TCTTTCCCTACCGACGCTCTTCCGATCT
A2(5’-3’)：GTGACTGGAGTCAGACGTGTGCTCTTCCGATCT
Recognition sequence has 12, is respectively ATCACG, TTAGGC, ACAGTG, TGACCA, GCCAAT, CAGATC, ACTTGA, ATCACG, TAGCTT, GGCTAC, CTTGTA, GATCAG, every section of recognition sequence can be added in F1 and R1 at random, and in F1 and R1, the array mode of recognition sequence can as the recognition signal of sample in sequencing result and gene.
2., for the primer of the outer qualitative detection of newborn infant's inherited metabolic disease genosome, it is characterized in that: as described in the form of at least 1 in table 1 ~ table 3;
The first round PCR primer sequence table of syndromic deafness is as following table 1; The first round PCR primer sequence table of G6PD gene is as following table 2; The sequence table of SLC12A3 gene first round PCR primer is as following table 3;
Table 1
Table 2
Table 3
3. test kit, is characterized in that: this test kit is containing, for example primer according to claim 2, and this test kit can detect multiple neonatal hereditary diease simultaneously.
4. test kit according to claim 4, is characterized in that:
May be used for detecting pku, argininemia, arginyluccinicaciduria, citrullinemia I type, citrullinemia II type, maple syrup urine disease, tyrosinemia I type, tyrosinemia II type, tyrosinemia type III, Ornithine carbamoyltransferase deficiency disease, galactosemia, glucose 6 phosphate dehydrogenase deficiency, isovaleric acidemia, propionic acidemia, glutaric acidemia I type, carnitine transit barrier, Carnitine palmitoyltransferase I deficiency disease, Carnitine palmitoyltransferase II deficiency disease, heredity nonsyndromic sensorineural, the gene that these 22 kinds of common neonatal hereditary diease of Gitelman syndrome are relevant.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861514A (en) * | 2016-05-27 | 2016-08-17 | 山东省立医院 | New mutant pathogenic gene SLC12A3 of Gitelman syndrome as well as encoded protein and application thereof |
| CN107058588A (en) * | 2017-06-09 | 2017-08-18 | 北京博奥医学检验所有限公司 | A kind of hereditary hearing impairment genetic test product |
| CN107937513A (en) * | 2017-11-30 | 2018-04-20 | 东莞市第八人民医院 | 50 kinds of hereditary disease genetic test probe groups of neonate and screening method |
| CN109097451A (en) * | 2018-04-23 | 2018-12-28 | 上海浦东解码生命科学研究院 | A kind of kit of detection G6PD deficiency disease Disease-causing gene mutation |
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| CN110468194A (en) * | 2019-08-12 | 2019-11-19 | 广州万德基因医学科技有限公司 | The multiple PCR primer group and kit in library are built for Inherited Metabolic Disorders high-flux sequence |
| CN110938685A (en) * | 2019-12-11 | 2020-03-31 | 福建福君基因生物科技有限公司 | Gene detection probe set for neonatal hereditary metabolic disease and hemoglobinopathy and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105861514A (en) * | 2016-05-27 | 2016-08-17 | 山东省立医院 | New mutant pathogenic gene SLC12A3 of Gitelman syndrome as well as encoded protein and application thereof |
| CN105861514B (en) * | 2016-05-27 | 2019-05-21 | 山东省立医院 | The new mutation Disease-causing gene SLC12A3 of Gitelman syndrome and its coding albumen and application |
| CN107058588A (en) * | 2017-06-09 | 2017-08-18 | 北京博奥医学检验所有限公司 | A kind of hereditary hearing impairment genetic test product |
| CN107937513A (en) * | 2017-11-30 | 2018-04-20 | 东莞市第八人民医院 | 50 kinds of hereditary disease genetic test probe groups of neonate and screening method |
| CN109097451A (en) * | 2018-04-23 | 2018-12-28 | 上海浦东解码生命科学研究院 | A kind of kit of detection G6PD deficiency disease Disease-causing gene mutation |
| CN110164504A (en) * | 2019-05-27 | 2019-08-23 | 复旦大学附属儿科医院 | Processing method, device and the electronic equipment of two generation sequencing datas |
| CN110164504B (en) * | 2019-05-27 | 2021-04-02 | 复旦大学附属儿科医院 | Method and device for processing next-generation sequencing data and electronic equipment |
| CN110172507A (en) * | 2019-06-11 | 2019-08-27 | 中国人民解放军第四军医大学 | A kind of Large Vestibular Aqueduct/Pendred syndrome Disease-causing gene SLC26A4 abrupt climatic change kit |
| CN110468194A (en) * | 2019-08-12 | 2019-11-19 | 广州万德基因医学科技有限公司 | The multiple PCR primer group and kit in library are built for Inherited Metabolic Disorders high-flux sequence |
| CN110938685A (en) * | 2019-12-11 | 2020-03-31 | 福建福君基因生物科技有限公司 | Gene detection probe set for neonatal hereditary metabolic disease and hemoglobinopathy and application thereof |
| CN114410771A (en) * | 2022-01-25 | 2022-04-29 | 广州盛安医学检验有限公司 | Primer library combination, kit and method for detecting neonatal genetic disease gene based on first-generation sequencing platform |
| CN114410771B (en) * | 2022-01-25 | 2023-10-03 | 广州盛安医学检验有限公司 | Primer library combination, kit and method for detecting neonatal genetic disease genes based on first-generation sequencing platform |
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